SUMMARY Endosomal Toll-like receptor (TLR) activation and late endosomal-initiated signaling are central mechanisms in innate immunity, inflammation and autoimmunity. Although nucleic acid-sensing endosomal TLR activation is important for a proper response to infection, unrestricted activation of endosomal TLRs initiates pro-inflammatory pathways that play a central role in the development of several disorders in humans including ischemia- reperfusion injury, rheumatoid arthritis, systemic lupus erythematosus, juvenile idiopathic arthritis and type 1 diabetes. Endosomal TLR activation requires the partial digestion of endosomal TLRs into their active forms, a process that depends on late endosome (LE) maturation. We have recently described a novel mechanism of late endosomal maturation in primary inflammatory cells that involves the direct binding of the calcium sensor Munc13-4 to the late endosomal SNARE protein syntaxin 7 (STX7), a regulator of membrane fusion. Calcium- dependent binding of Munc13-4 to STX7 regulates endosomal maturation and TLR signaling. Importantly, the late endosomal defective phenotype observed in Munc13-4-deficient cells is rescued by wild type Munc13-4 but not by a calcium-binding-deficient Munc13-4 mutant that impairs the STX7-Munc13-4 interaction. Our data identify the interaction of Munc13-4 with syntaxin 7 as an essential process for the regulation of endosomal TLR activation. We propose that interference with the interaction of Munc13-4 with STX7 prevents late endosomal maturation and decreases inflammation by impairing TLR7 and TLR9-dependent signaling pathways. This is supported by our preliminary data showing that the inflammatory response to in vivo challenge with endosomal TLR9 ligands but not with TLR ligands that operate through plasma membrane receptors is decreased in Munc13-4-deficient mice. The objective of this proposal is to utilize high-throughput screening to identify small- molecule inhibitors of the complex formed by Munc13-4 and syntaxin 7 for use in primary immune cells that contribute to systemic inflammation. We also aim to validate these compounds through established secondary assays and cell-based approaches. Our specific Aims are: 1) To utilize high-throughput screening for small- molecule inhibitors of syntaxin 7-Munc13-4 binding using an innovative approach that analyzes the activation of the complex on intact intracellular endosomes; 2) To perform orthogonal confirmation assays, cell-based secondary approaches and analysis of the molecular similarity of the active series to identify and prioritize active probes and 3) To validate active probes using analysis of mechanisms of endosomal maturation and nucleic acid-sensing TLR-initiated signaling in primary immune cells. The significance of the research proposed is that new small-molecule inhibitors that selectively and specifically inhibit the syntaxin 7-Munc13-4 complex and nucleic acid-sensing TLR signaling, will lead to the development of novel pre-therapeutic leads for the treatment of diseases in which systemic inflammation is upregulated including autoimmune diseases.